Compositions and methods for using engineered deubiquitinases for probing ubiquitin-dependent cellular processes

Abstract

The present disclosure provides, inter alia, a recombinant engineered deubiquitinase (DUB) and methods for treating or ameliorating an inherited ion channelopathy, such as long QT syndrome, Brugada syndrome, or cystic fibrosis, in a subject. Further provided are methods for screening mutations causing such inherited ion channelopathies for a trafficking-deficient mutation that is treatable by the recombinant engineered DUB disclosed herein.

What is claimed is: 
     
       1. An RNA molecule encoding a recombinant engineered deubiquitinase (DUB) comprising:
 a) a catalytic unit comprising the catalytic domain of a deubiquitinase; 
 b) a protein binder comprising an antibody, or antigen binding fragment thereof, that specifically binds a target substrate protein for deubiquitination by the engineered DUB; and 
 c) a variable linker between the catalytic unit and the protein binder. 
 
     
     
       2. The RNA molecule of  claim 1 , wherein the RNA molecule is an mRNA molecule. 
     
     
       3. The RNA molecule of  claim 1 , wherein the catalytic unit is selective for a particular ubiquitin linkage type. 
     
     
       4. The RNA molecule of  claim 1 , wherein the catalytic unit comprises the catalytic domain of a deubiquitinase, wherein the deubiquitinase is from the ubiquitin specific proteases (USP) family, the ovarian tumor proteases (OTU) family, the ubiquitin C-terminal hydrolases (UCH) family, the Josephin domain (Josephin) family, the motif interacting with ubiquitin-containing novel DUB (MINDY) family, or the JAB1/MPN/Mov34 metalloenzyme domain (JAMM) family. 
     
     
       5. The RNA molecule of  claim 1 , wherein the catalytic unit comprises the catalytic domain of a deubiquitinase from the USP family. 
     
     
       6. The RNA molecule of  claim 1 , wherein the catalytic unit comprises the catalytic domain of USP21. 
     
     
       7. The RNA molecule of  claim 1 , wherein the catalytic unit comprises the catalytic domain of a deubiquitinase from the OTU family. 
     
     
       8. The RNA molecule of  claim 1 , wherein the catalytic unit comprises the catalytic domain of OTUD1. 
     
     
       9. The RNA molecule of  claim 1 , wherein the catalytic unit comprises the catalytic domain of OTUD4. 
     
     
       10. The RNA molecule of  claim 1 , wherein the catalytic unit comprises the catalytic domain of Cezanne. 
     
     
       11. The RNA molecule of  claim 1 , wherein the catalytic unit comprises the catalytic domain of TRABID or the catalytic domain of OTULIN. 
     
     
       12. The RNA molecule of  claim 1 , wherein the catalytic unit comprises the catalytic domain of USP21 and is capable of non-selectively eliminating all ubiquitin linkage types. 
     
     
       13. The RNA molecule of  claim 1 , wherein the catalytic unit comprises the catalytic domain of OTUD1 and is capable of selectively eliminating K63 ubiquitin linkages; or the catalytic domain of OTUD4 and is capable of selectively eliminating K48 ubiquitin linkages. 
     
     
       14. The RNA molecule of  claim 1 , wherein the catalytic unit comprises the catalytic domain of Cezanne and is capable of selectively eliminating K11 ubiquitin linkages. 
     
     
       15. The RNA molecule of  claim 1 , wherein the catalytic unit comprises the catalytic domain of TRABID and is capable of selectively eliminating K29 ubiquitin linkages, K33 ubiquitin linkages, or K29 and K33 ubiquitin linkages; or the catalytic domain of OTULIN and is capable of selectively eliminating Met1 ubiquitin linkages. 
     
     
       16. The RNA molecule of  claim 1 , wherein the catalytic unit is non-selective for a particular ubiquitin linkage type. 
     
     
       17. The RNA molecule of  claim 1 , wherein the antibody is a nanobody, scFv, (scFv)2, Fab, Fab′, F(ab′)2, Fv, diabody, or a DARPin. 
     
     
       18. The RNA molecule of  claim 1 , wherein the antibody is a scFv, (scFv)2, Fab, Fab′, F(ab′)2, Fv, antibody mimetic, or diabody. 
     
     
       19. The RNA molecule of  claim 1 , wherein the antibody is a single domain antibody (dAb). 
     
     
       20. The RNA molecule of  claim 1 , wherein the antibody is a nanobody. 
     
     
       21. The RNA molecule of  claim 1 , wherein the catalytic unit comprises the catalytic domain of Cezanne and the antibody is a nanobody. 
     
     
       22. The RNA molecule of  claim 1 , wherein the catalytic unit comprises the catalytic domain of Cezanne and the antibody is a single domain antibody (dAb). 
     
     
       23. The RNA molecule of  claim 2 , wherein the antibody is a nanobody, scFv, (scFv)2, Fab, Fab′, F(ab′)2, Fv, diabody, or a DARPin. 
     
     
       24. The RNA molecule of  claim 2 , wherein the antibody is a scFv, (scFv)2, Fab, Fab′, F(ab′)2, Fv, antibody mimetic, or diabody. 
     
     
       25. The RNA molecule of  claim 2 , wherein the antibody is a single domain antibody (dAb). 
     
     
       26. The RNA molecule of  claim 2 , wherein the antibody is a nanobody. 
     
     
       27. The RNA molecule of  claim 2 , wherein the catalytic unit comprises the catalytic domain of Cezanne and the antibody is a nanobody. 
     
     
       28. The RNA molecule of  claim 2 , wherein the catalytic unit comprises the catalytic domain of Cezanne and the antibody is a single domain antibody (dAb). 
     
     
       29. The RNA molecule of  claim 1 , wherein the protein binder specifically binds an ion channel. 
     
     
       30. The RNA molecule of  claim 29 , wherein the ion channel is KCNQ1, HERG, or CFTR.